U.S. patent application number 11/293489 was filed with the patent office on 2006-05-04 for automated molecular pathology apparatus having fixed slide platforms.
Invention is credited to Charles D. Lemme, William Richards, Wayne Showalter.
Application Number | 20060093520 11/293489 |
Document ID | / |
Family ID | 38481740 |
Filed Date | 2006-05-04 |
United States Patent
Application |
20060093520 |
Kind Code |
A1 |
Lemme; Charles D. ; et
al. |
May 4, 2006 |
Automated molecular pathology apparatus having fixed slide
platforms
Abstract
Apparatus and methods for automatically staining or treating
multiple tissue samples mounted on slides are provided, in which
the slides and reagent bottles are held in fixed position, and the
reagent and wash solutions brought to the slides.
Inventors: |
Lemme; Charles D.; (Tucson,
AZ) ; Richards; William; (Tucson, AZ) ;
Showalter; Wayne; (Tucson, AZ) |
Correspondence
Address: |
HAYES, SOLOWAY P.C.
3450 E. SUNRISE DRIVE, SUITE 140
TUCSON
AZ
85718
US
|
Family ID: |
38481740 |
Appl. No.: |
11/293489 |
Filed: |
December 2, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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10283639 |
Oct 30, 2002 |
|
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|
11293489 |
Dec 2, 2005 |
|
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60350273 |
Nov 2, 2001 |
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Current U.S.
Class: |
422/64 ;
422/65 |
Current CPC
Class: |
G01N 1/312 20130101;
Y10T 436/112499 20150115; Y10T 436/2575 20150115 |
Class at
Publication: |
422/064 ;
422/065 |
International
Class: |
G01N 35/00 20060101
G01N035/00 |
Claims
1-47. (canceled)
48. A method for processing a slide having a biological sample
thereon comprising the steps of: (a) preparing a slide for
biological treatment with a reagent; (b) moving a reagent dispenser
to a selected reagent bottle; (c) aspirating a pre-selected volume
of reagent from the reagent bottle; (d) moving the reagent
dispenser to the slide; (e) dispensing the pre-selected volume of
reagent onto the slide; (f) preparing a second slide for treatment
with a reagent; (g) cleaning the reagent dispenser; (h) moving a
reagent dispenser to a selected reagent which may be the same or
different from the reagent of step (b); (i) aspirating a
pre-selected amount of reagent selected in the preceding step to
the reagent dispenser means; (j) moving the reagent dispenser means
to said second slide; (k) dispensing said pre-selected volume of
reagent onto said second slide; (l) cleaning the reagent dispensing
means; and (m) repeating steps (h)-(l).
49. The method of claim 48, wherein two reagent dispensers are
provided, and including the step of cleaning one of the reagent
dispensers while the other reagent dispenser is employed to
dispense reagent onto a selected slide.
50. The method according to claim 48, wherein said reagent
dispenser comprises a robotic syringe, and reagent is taken up by
aspiration.
51. The method according to claim 48, including the step of heating
the slide before, during or following treatment with a reagent.
52. The method according to claim 48, including the step of
applying a cleaning reagent or wash solution onto said slide.
53. The method according to claim 52, including the step of heating
the slide before, during or following application of said cleaning
reagent or wash solution.
54. The method according to claim 48, including the step of
leveling and/or stripping reagent from the slide by means of an air
jet or air knife.
55. The method according to claim 48, including the step of
applying a cover fluid to the slide.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority for U.S. Provisional
Application Ser. No. 60/350,273, filed Nov. 2, 2001.
FIELD OF THE INVENTION
[0002] The present invention is directed to apparatus for use in
diagnostic molecular pathology and, more particularly, to such
apparatus used for the automated staining and/or treating of tissue
samples mounted on microscope slides.
BACKGROUND OF THE INVENTION
[0003] Molecular pathology is the examination at a molecular level
of the DNA, mRNA, and proteins that cause or are otherwise
associated with disease. From this examination important
information about patient diagnosis, prognosis, and treatment
options can be elucidated. The practice of molecular pathology is
generally divided into two main areas: (i) analysis of DNA, mRNA,
and proteins in intact cells (in-situ), and (ii) analysis of these
biological materials after they have been extracted from tissues.
The first category, to which the present invention is primarily
directed, has the advantage that it allows the pathologist or
scientist to study the histopathologic architecture or morphology
of the tissue specimen under the microscope at the same time that
the nucleic acid or proteins are being assayed. These techniques
include immunohistochemistry (IHC) which looks at proteins, in-situ
hybridization (ISH) which looks at nucleic acids, histochemistry
(HC) which looks at carbohydrates, and enzyme histochemistry (EHC)
which looks at enzyme chemistry. For example, ISH can be used to
look for the presence of a genetic abnormality or condition such as
amplification of cancer causing genes specifically in cells that,
when viewed under a microscope, morphologically appear to be
malignant. ISH is also useful in the diagnosis of infectious
diseases as it allows detection not only of a microbial sequence
but also of precisely which cells are infected. This may have
important clinicopathologic implications and is an effective means
to rule out the possibility that positive hybridization signal may
have come from an adjacent tissue of no clinical concern or from
blood or outside contamination.
[0004] IHC utilizes antibodies which bind specifically with unique
epitopes present only in certain types of diseased cellular tissue.
IHC requires a series of treatment steps conducted on a tissue
section or cells (e.g. blood or bone marrow) mounted on a glass
slide to highlight by selective staining certain morphological
indicators of disease states. Typical steps include pretreatment of
the tissue section to remove the paraffin and reduce non-specific
binding, retrieval of antigens masked by cross-linking of the
proteins from the chemical fixatives, antibody treatment and
incubation, enzyme labeled secondary antibody treatment and
incubation, substrate reaction with the enzyme to produce a
fluorophore or chromophore highlighting areas of the tissue section
having epitopes binding with the antibody, counterstaining, and the
like. Most of these steps are separated by multiple rinse steps to
remove unreacted residual reagent from the prior step. Incubations
can be conducted at elevated temperatures, usually around
37.degree. C., and the tissue must be continuously protected from
dehydration. ISH analysis, which relies upon the specific binding
affinity of probes with unique or repetitive nucleotide sequences
from the cells of tissue samples or bodily fluids, requires a
similar series of process steps with many different reagents and is
further complicated by varying temperature requirements.
[0005] In view of the large number of repetitive treatment steps
needed for both IHC and ISH, automated systems have been introduced
to reduce human labor and the costs and error rate associated
therewith, and to introduce uniformity. Examples of automated
systems that have been successfully employed include the ES.RTM.,
NexES.RTM., DISCOVERY.TM., BENCHMARK.TM. and Gen II.RTM. staining
Systems available from Ventana Medical Systems (Tucson, Ariz.).
These systems employ a microprocessor controlled system including a
revolving carousel supporting radially positioned slides. A stepper
motor rotates the carousel placing each slide under one of a series
of reagent dispensers positioned above the slides. Bar codes on the
slides and reagent dispensers permits the computer controlled
positioning of the dispensers and slides so that different reagent
treatments can be performed for each of the various tissue samples
by appropriate programming of the computer.
[0006] Instrumentation such as the Ventana Medical Systems ES.RTM.,
NexES.RTM., BENCHMARK.RTM. and DISCOVERY.RTM. systems are
fundamentally designed to sequentially apply reagents to tissue
sections mounted on one by three inch glass microscope slides under
controlled environmental conditions. The instrument must perform
several basic functions such as reagent application, washing (to
remove a previously applied reagent), jet draining (a technique to
reduce the residual buffer volume on a slide subsequent to
washing), Liquid Coverslip.TM. application (a light oil application
used to contain reagents and prevent evaporation), and other
instrument functions.
[0007] The Ventana Medical Systems staining instruments mentioned
above process slides on a rotating carousel. The instrumentation
described herein has the slides fixed in a stationary position and
rotates the basic processing stations above the fixed slides. The
following details of how the slides are processed, the process
algorithm, is the same regardless of the physical
configuration.
[0008] The process of staining tissue on a slide consists of the
sequential repetition of the basic instrument functions described
above. Essentially a reagent is applied to the tissue then
incubated for a specified time at a specific temperature. When the
incubation time is completed the reagent is washed off the slide
and the next reagent is applied, incubated, and washed off, etc,
until all of the reagents have been applied and the staining
process is complete.
[0009] It is desirable to permit any staining protocol for any of
the slides being run, i.e. any combination of reagents and
incubation times. In addition, to stain multiple slides as quickly
as possible the instrument should process the slides
simultaneously. This is feasible given that most of the time slides
are just incubating, thus freeing up time to perform the washing,
reagent application and other functions on other slides.
[0010] One algorithm to accomplish simultaneous staining (sometimes
referred to as the "random access" method) is to create a task and
time schedule for each slide in the run, then perform each task on
each slide when the schedule calls for it. The problem with this
method is that incubation times will not be accurate if the
instrument is busy performing a task on one slide when it is time
to be washing another slide (thereby completing incubation on that
slide). The variation in incubation times will be unpredictable
since the total number of slides and the slide protocols vary.
[0011] Slide processing using the lock step algorithm insures that
all incubation times are -accurate and predictable irrespective of
the number of slides processed or the variation in slide protocols.
While incubation times are assured, the lock step algorithm implies
that incubation times must be an increment of the fundamental
incubation time period. In the above example the incubation period
is two minutes, therefore total incubation times must be two, four,
six, eight etc. minutes in duration. However, the preferred
embodiment of the present invention uses a four minute incubation
time. Generally this is not a particular limitation since typical
incubation times are an order of magnitude longer than the
fundamental incubation period.
[0012] Prior art staining systems typically include either
convection or radiation to warm the samples above laboratory
ambient temperatures for steps requiring elevated temperatures.
Heating the slide improves staining quality by acceleration of the
chemical reaction and can permit a reaction temperature more
closely matching body temperature (about 37.degree. C.) at which
antibodies are designed to react. While such convection or radiant
heating systems have been generally suitable for IHC, which is
antibody based, they are less suitable for ISH, which is nucleic
acid based and requires higher and more precise temperature
control. In order to denature the DNA double helix of both the
target sample and the probe so as to render them single stranded,
the temperature must be raised above the melting point of the
duplex, usually about 94.degree. C. Precise temperature control is
also required in ISH to effect probe hybridization at the desired
stringency. The selected temperature must be low enough to enable
hybridization between probe and target, but high enough to prevent
mismatched hybrids from forming.
[0013] Hot air convection, conduction or radiant heat heating units
typically employed with prior art automated tissue stainers do not
permit the temperature of individual slides to be separately
controlled. With prior art systems all of the slides are heated to
the same temperature at any given time during the process. For
example, U.S. Pat. No. 5,645,114 to Bogen et al. discloses a
dispensing assembly adapted to carry a plurality of microscope
slides. Individual slide holders containing resistive heating units
are provided. However, with the assembly taught by Bogen et al.,
all of the slides would be heated to a common temperature because
no means are disclosed for separate heating controls or for
shielding slides from heat generated by adjacent slides.
[0014] Other difficulties frequently encountered in both IHC and
ISH testing results from the manner in which the tissues are
typically preserved. The mainstay of the diagnostic pathology
laboratory has been for many decades the formalin-fixed, paraffin
embedded block of tissue, sectioned and mounted upon glass slides.
Fixation in such a preservative causes cross-linking of
macromolecules, both amino acids and nucleic acids. These
cross-linked components must be removed to allow access of the
probe to the target nucleic acid and to allow the antibody to
recognize the corresponding antigen. "Unmasking" the antigen and/or
nucleic acid is typically accomplished manually with multiple
pretreatment, protolytic digestion, and wash steps.
[0015] Prior to staining, complete removal of the paraffin is also
required so that it does not interfere with antibody or probe
binding. Deparaffinization normally is achieved by the use of two
or three successive clearing reagents that are paraffin solvents
such as xylene, xylene substitutes or toluene.
[0016] The foregoing discussion of the prior art largely derives
from Richards et al. U.S. Pat. No. 6,296,809, assigned to Ventana
Medical Systems, in which there is described apparatus and methods
for automatically staining or treating multiple tissue samples
mounted on microscope slides so that each sample can receive an
individualized staining or treatment protocol even when such
protocols require different temperature parameters. More
specifically, there is described in the '809 patent apparatus
comprising a computer controlled, bar code driven, staining
instrument that automatically applies chemical and biological
reagents to tissue or cells mounted or affixed to standard glass
microscope slides. According to the '809 patent, a plurality of
slides are mounted in a circular array on a carousel which rotates,
as directed by the computer, to a dispensing location placing each
slide under one of a series of reagent dispensers on a second
rotating carousel positioned above the slides. Each slide receives
the selected reagents (e.g. DNA probe) and is washed, mixed and/or
heated in an optimum sequence and for the required period of
time.
[0017] According to the '809 patent, individual slides are carried
on thermal platforms radially mounted to the carousel. Sensors also
mounted to the slide carousel, individually monitor and control
each thermal platform separately. Apparatus made in accordance with
the '809 patent is available commercially from Ventana Medical
Systems, of Tucson, Ariz. as the DISCOVERY.TM. or BENCHMARK.TM.
systems.
[0018] The present invention is a modification and improvement over
the prior art including the apparatus and methods described in the
'809 patent. More particularly, the present invention rather than
bringing the slides to the reagent, stain, and wash stations,
brings the reagent, stain and wash stations to fixedly positioned
slides. That is to say, in the present invention the slides are
fixedly positioned in the apparatus, and the various washing,
staining and reagent fluids selectively delivered to the slides.
Fixing the slides in position in the apparatus eliminates expensive
and disposable dispensers, and simplifies wiring to the heaters,
and also eliminates the potential that a slide may be dislocated by
rapid start and stop movement of the slide carousel, which, in a
worst case scenario could result in a domino or train-wreck effect
where one dislocated slide hits the neighboring slide causing that
slide to dislocate, and so forth. Additionally, maintaining the
slides in fixed position eliminates inertial problems of a
high-volume reagent and slide carousel. Thus, motors and bearings
need not be so robust.
[0019] Further features and advantages of the present invention
will be seen from the following detailed description, taken in
conjunction with the accompanying drawings, wherein like numerals
depict like parts, and wherein:
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] FIG. 1 is a perspective view of the apparatus of the present
invention shown with the slide cabinet shell removed;
[0021] FIG. 2 is a perspective view of the apparatus of the present
invention shown in conjunction with a computer and other
instruments with which it operates;
[0022] FIG. 3 is a perspective view and FIG. 3a is an exploded view
of details of the nozzle plate portion of the present
invention;
[0023] FIG. 4 is an exploded view of details of the slide plate
portion of the present invention;
[0024] FIGS. 5 and 6 are perspective views, from the top and the
bottom, respectively, of portions of the slide plate portion of the
present invention;
[0025] FIG. 7 is a perspective view of the reagent plate portion of
the present invention;
[0026] FIG. 8 is a perspective view showing two reagent bottles of
the present invention;
[0027] FIG. 9 is a top plan view of two reagent bottles of the
present invention; and
[0028] FIG. 10 is a flow chart of the operation and control of the
present invention.
DETAILED DESCRIPTION OF THE INVENTION
[0029] Referring now in detail to the drawings wherein like parts
are designated by like reference numerals throughout, there is
illustrated in FIG. 1 a perspective view of the molecular pathology
apparatus according to the present invention which is designated
generally by reference numeral 10. For the purposes of clarity,
several of the reagent bottles, as well as the cabinet shell, and
liquid and air supply tubing and electrical wiring are omitted from
the drawings. Apparatus 10 is designed to automatically stain or
otherwise treat tissue mounted on microscope slides with nucleic
acid probes, antibodies, and/or other reagents in a desired
sequence, time and temperature. Tissue sections so stained or
treated are then to be viewed under a microscope by a medical
practitioner who reads the slide for purposes of patient diagnosis,
prognosis, or treatment selection.
[0030] In a preferred embodiment, apparatus 10 functions as one
component or module of a system 12 (FIG. 2) which also comprises a
host computer 14 preferably a personal computer, monitor 16,
keyboard 18, mouse 20, bulk fluid containers 22, waste container 23
and related equipment. Additional staining modules or other
instruments may be added to system 12 to form a network with
computer 14 functioning as a server. Alternatively, some or all of
these separate components could be incorporated into apparatus 10
making it a stand-alone instrument. Referring also to FIGS. 3-7, as
set forth in greater detail below, a plurality of slide platforms
50 are mounted radially about a center point 32 of drawer 34 upon
which standard glass slides 60 with tissue samples may be placed.
Drawer 34 is preferably constructed of stainless steel and is
slidably mounted in housing 30 on rails 40 or the like. The
temperature of each slide may be individually controlled by means
of sensors and a microprocessor, i.e. as taught in the
above-mentioned '809 patent.
[0031] Each of the slide platforms 50 is connected through
individual wires and a wiring harness (not shown) to a
microprocessor. A feature and advantage of the present invention
which results from fixedly mounting the slide platforms in drawer
34 is that each of the heaters and thermal sensors may be hardwired
thereby eliminating the need for a slip ring assembly or rotor
couplings, as well as complex stepping motors, etc. for locating
and positioning a rotating slide carousel as required in prior art
devices. Also, the possibility that a slide or slides may be
shifted or dislocated during rapid start and stop rotation of the
slide carousel is eliminated.
[0032] In a particularly preferred embodiment, a plurality of slots
or channels are formed on the top surface of each of the slide
heaters, i.e. the interface surface between the slide heater and
the slide, for gathering and venting gas bubbles as may form during
heating, i.e. in accordance with co-pending U.S. application Ser.
No. 09/953,417, filed Sep. 11, 2001, and assigned to the common
assignee, which disclosure is incorporated herein by reference.
[0033] Referring also to FIGS. 1, 2, 5 and 6, drawer 34 includes a
circular pan 35 having a peripherial wall 36 serving as a splash
guard, a peripheral trough 37 and a central drain 38, i.e. at
center point 32, both connected to drain lines (39) which in turn
are connected to waste container 23. Drawer 34 is slidably mounted
in housing 30 on rails 40. Rails 40, in a preferred embodiment,
comprise three piece telescoping rails so that the drawer 34 may be
slid clear of housing 30 to permit access to all of the slide
platforms 50 for slide loading and removal. A damping means 42 such
as a pneumatic means, electromotive means, mechanical spring damper
or the like preferably is provided to smooth movement of the drawer
whereby to avoid possible dislodging of slides, particularly when
the drawer is closed. Also, in a preferred embodiment of the
invention, rails 40 are supported on a lift mechanism such as
pneumatic cylinders 52 (see FIG. 1), which automatically index to
permit the rails 40 to move up and down so that the drawer 34 may
be dropped to permit wall 36 to clear the nozzle plate 100 when the
drawer is slid in and out of the apparatus.
[0034] Slide drawer 34 is divided into thirty-five equal pie-shaped
sections 70. Thirty of the pie-shaped sections 70 are occupied by
slide platforms 50 while the five remaining pie-shaped sections 70A
(FIG. 4) at the rear of the drawer are devoid of slide platforms
50. In other words, a row of thirty slide platforms 50 are radially
mounted on drawer 34 and evenly spaced from one another, except at
the ends of the row.
[0035] However, the invention is not limited to thirty active slide
locations, and more or fewer slide locations may be employed. An
alternative embodiment may be implemented by aligning the platforms
50 linearly, which results in potentially limitless number of
platforms.
[0036] Referring to FIGS.1 and 3, a nozzle plate 100 is
concentrically and rotatably mounted above slide drawer 34. Nozzle
plate 100 is mounted on a shaft (not shown) supported by a bridge
110, and driven by a computer controlled stepping motor and drive
belt (not shown), and rotates 185.degree. plus or minus from a home
position 104 at the rear of the drawer. The computer controlled
stepper motor and drive belt are conventional in this art.
Accordingly, details are omitted for the sake of clarity.
[0037] Nozzle plate 100 carries the various slide treatment
stations, other than the reagent dispensing station. Thus, nozzle
plate 100 carries dual rinse nozzle block 102, volume
adjust/stringency block 103, Liquid Coverslip.TM. evaporation
inhibitor liquid application block 104, vortex mixer air jet block
106, jet drain knives 108, and the like, all for preparing a slide
for staining, stain removal, and the like, and to clear bar codes
110 carried on the slides, and a bar code reader 109, all as
described in detail in U.S. Pat. No. 5,654,200 to Copeland et al,
which disclosure is incorporated herein by reference. In other
words, nozzle plate 100 carries all of the functions for slide
preparation, cleaning, reagent mixing, Liquid Coverslip.TM.
application, etc. other than reagent application, as described in
the '200 patent to Copeland et al., plus wash stations 121, 122 for
the reagent application probes as will be described in detail
below.
[0038] Preferably, but not necessarily, the various rinse nozzle
blocks, vortex mixer air jet blocks, air knives, etc. are arranged
adjacent to one another so that the nozzle plate may be indexed and
advanced in a "lock-step" manner to sequentially treat a slide
according to an accepted protocol. For example, air knives 108 may
be arranged immediately adjacent rinse nozzle blocks 106 so that
nozzle plate 100 may be advanced in "lock step" manner past a
selected slide, and the slide rinsed and fluid stripped, etc. Also,
if desired, vortex mixer airjet blocks 106 may be oriented to
impinge simultaneously on two adjacent slides.
[0039] For the sake of clarity, fluid and air supply tubing for the
several slide treatment stations have been omitted from the
drawings. It will be understood, however, that the fluid and air
supply tubing are made long enough to permit 185.degree. rotation
of the valve plate so that each slide treatment station can reach
each slide 60. A pair of wash stations 121, 122 spaced two
thirty-fifths of a revolution (approximately 20.572.degree.) apart
as will be described in detail hereinafter, are also attached to
and radially extend beyond the periphery of the nozzle plate 100,
and rotate with the nozzle plate 100.
[0040] Reagent plate 300 is fixedly mounted to arch 110 vertically
above nozzle plate 100, which arch in turn is fixedly mounted
within housing 30. A plurality of reagent bottles 302 are removably
mounted within recesses 304 formed equally spaced adjacent the
periphery of reagent plate 300. In the illustrated embodiment, a
total of thirty-five reagent bottles are mounted on the reagent
plate, spaced approximately one thirty-fifth (approximately
10.286.degree.) apart.
[0041] The reagents may include any chemical or biological material
conventionally applied to slides including nucleic acid probes or
primers, polymerase, primary and secondary antibodies, digestion
enzymes, pre-fixatives, post-fixatives, readout chemistry,
counterstains, and the like.
[0042] Referring also to FIG. 8, the reagent bottles 302 each
comprise a cylindrical hollow body 305 closed at the bottom end by
an integrally formed bottom wall (not shown). Each bottle 302
includes an integrally formed bracket 306 which serves to maintain
the bottles 304 at a desired height in reagent plate 300, and which
serves also to permit the stringing together of a plurality of like
bottles 302. Accordingly, each bracket 302 includes a hinge element
308 for cooperating with a hinge element 310 of an adjacent bottle
302. In the illustrated embodiment, hinge elements 308 and 310 are
shown as conventional pin-hinges in which the upper hinge 308
includes a pin 312 which fits into the lower hinge 310, i.e.
similar to a conventional door hinge. However, bottles 302 may be
hinged together in a variety of ways.
[0043] Bracket 306 preferably includes a flat surface 314 upon
which is carried a bar code 316 for identifying the contents of the
bottle 302. Bottles 302 also include an insert 318 having a tapered
top surface 320 fitted in the top end of the bottles for locating a
reagent transfer probe as will be described in detail hereinafter,
and a cap 322 which may be either twist or snap-fitted to the
bottle 302 for sealing the bottle 302.
[0044] Making brackets 306 attachable to one another permits a lab
worker to assemble a chain of reagents for use, and also to remove
the chain of reagents so that the reagents may be refrigerated, for
example, overnight when not in use.
[0045] Referring next to FIG. 9, the side walls 324 of brackets 306
are tapered so that a pie-shaped space 326 is formed between two
bottles when two bottles are fastened together in a string, and
mounted in recesses 104 in the reagent plate 300, thereby exposing
holes 328 formed through reagent plate 300. Holes 328 are formed in
the same concentric circle as recesses 304, and are spaced
equidistant between adjacent recesses 304. The purpose of
pie-shaped spaces 326 and holes 328 is to provide clearance for
reagent transfer probes 402, 404 as will be described in detail
below.
[0046] Referring again to FIG. 1, an arm 400 is rotatably mounted
on arch 405 concentrically above reagent plate 300, and carries a
pair of reagent transfer probes 402, 404 located at the distal end
of arm 400 and spaced approximately 10.286.degree. apart. Arm 400
also carries a bar code reader 406 for reading bar codes 316 on the
reagent bottles. Arm 400 is rotatably driven by a computer driven
stepping motor (not shown), and rotates plus or minus 185.degree.
in either direction from a home position 410.
[0047] Reagent transfer probes 402 and 404, which are identical to
one another, preferably comprise automatic pipette
metering/dispensing pick-up devices designed to aspirate or "sip"
reagent from a reagent bottle, move to a slide, and then "spit" or
deposit the reagent onto the slide. "Sip" and "spit" automatic
pipette/metering dispensing pick-up devices are described in
published PCT Application No. PCT/US99/04379, which disclosure is
incorporated herein by reference. Reagent transfer probes 402 and
404 are carried on the distal end of arm 400 and are spaced from
one another so that when one of the probes, e.g. probe 402 is
located centrally over a slide 60, the other reagent transfer probe
404 may be centrally positioned over one of the two probe wash
stations 121 or 122. A pneumatic cylinder (not shown) selectively
raises and lowers probes 402 and 404 into one of the following
positions: a raised transport position above the tops of the
bottles 302 where the arm 400 is free to rotate; a reagent drawing
position wherein one of the probes is inserted into a selected
reagent bottle 302 wherein a measured amount of reagent may be
drawn into the probe; a reagent dispensing position wherein a
reagent transfer probe containing reagent is disposed in the
pie-shaped space 326 between two reagent bottles, above a selected
slide to dispense reagent thereon; and a cleaning position wherein
the other probe, i.e. the probe not being used to dispense reagent,
is operatively disposed in one of probe washing stations 121 or
122. While the apparatus of the present invention could be made
with only a single reagent transfer probe, providing two spaced
reagent transfer probes essentially doubles cycle speed since
reagent metering may be accomplished using one of the two reagent
transfer probes while the other of the two reagent transfer probes
is going through the wash cycle as will be described below. That is
to say, while one of the reagent transfer probes, e.g. reagent
transfer probe 402 is dispensing reagent onto a slide, the other
reagent transfer probe, i.e. idle reagent transfer probe 404 may be
lowered to a probe wash station 121 where the idle reagent transfer
probe may be rinsed inside and out at the same time.
[0048] Referring to FIG. 10, the overall process is as follows:
[0049] A plurality of specimen-bearing slides 60 are mounted on the
slide platforms 50, selected reagent bottles 302 mounted in the
reagent plate, the slide drawer is closed and the slide bar codes
are read. The computer than downloads the run steps for the entire
run, the nozzle plate 100 is indexed to the first slide, and the
slide is washed and prepared for staining or other treatment in
accordance with the pre-programmed run steps by advancing the
nozzle plate in "lock-step" manner. In the meanwhile, probe arm 400
is rotated to the appropriate reagent bottle 302, one of the two
reagent transfer probes 402 or 404 is indexed over the selected
reagent bottle, and the probe lowered to aspirate a measured amount
of the desired reagent. The reagent-containing transfer probe is
then raised, and the arm 400 moved to the selected slide where the
loaded reagent transfer probe is lowered to just over the slide,
and the reagent dispensed on the slide. In the meanwhile, the idle
reagent transfer probe is lowered into one of the washing stations
121 or 122, wherein the reagent transfer probe is washed inside and
out. Both reagent transfer probes 402 and 404 are then raised, and
the process repeated, but using the reagent transfer probe just
cleaned in the previous step to aspirate and dispense reagent onto
the next slide. As before, simultaneously with dispensing the
reagent onto the slide as in the previous case, the idle reagent
transfer probe is washed while the active reagent transfer probe is
dispensing reagent onto the new slide.
[0050] The foregoing steps are repeated until all of the slides are
processed. For convenience, in the illustrated embodiment, the
dwell time at each slide station is approximately six and
two-thirds seconds. This comes from dividing a four minute cycle
time into thirty-six time spaces, one time space for each of the
thirty slide positions plus five blank slide positions, plus one
"virtual" time space for returning the arm 400 from the last slide
position to the first slide position. The virtual slide position
allows the nozzle plate to return to the other end of its travel
range in an uninterrupted fashion.
[0051] The staining algorithm used on the aforesaid Ventana systems
avoids the above problem by using a "lock step" method. The lock
step algorithm requires that the nozzle plate which holds the
processing functions be rotated one slide position index every n
seconds, termed the slide index time. The slide index time is
preferably as short as possible but long enough that the function
that requires the longest time can be completed within the index
time. Index times are usually on the order of several seconds. The
time for one complete rotation of the nozzle plate, termed the
fundamental incubation period, will then be n times the number of
slide positions. (For example, if the slide index time is six
seconds and there are twenty slide positions, the incubation time
period will be 120 seconds or two minutes.)
[0052] Throughout the entire run the nozzle plate is indexed one
slide position every n seconds. After the index, the system checks
the schedule to see if any of the slides at each of the processing
stations require the function of that, station. For example, if the
slide at the washing station is scheduled for washing, that slide
is washed. Similarly if the slide at the reagent application
station is scheduled for the application of a new reagent, then the
new reagent is applied.
[0053] The above-described invention has several advantages over
the prior art. For one, making the slide plate fixed in position
reduces the possibility of a slide being dislocated during the
rapid start-stop rotational movement of a slide carousel. Also,
employing two transfer syringes insures better cleaning of transfer
syringes without increasing cycle time. Also, using vials or
bottles for reagents eliminates the prior art's reliance on complex
and costly dispensers.
[0054] Also, since none of the moving elements, i.e. nozzle plate
100 and probe support arm 400 need travel more than plus or minus
185.degree. in either direction, all electrical connections, and
air and fluid-connections can be achieved without the need for slip
ring or rotary connections, since the hoses and wires are quite
capable of taking twistings of 185.degree. plus.
[0055] The instrumentation described herein may or may not have the
ability to continuously rotate the nozzle plate. The nozzle plate
may need to return to a starting position before rotation has
exceeded 360 degrees. This may also be required when the slides are
rotated on a carousel and the processing functions are fixed above
the slides. Similarly, other non rotating designs are possible such
as linear or two dimensional configurations. In these cases there
will be a requirement to move the slides or processing functions
back to the original starting position during the staining run. In
most cases it is likely that the time required to do this will
exceed the index time which violates the fundamental requirement of
the lock step algorithm. The lock step algorithm can still be used
by introducing the concept of a "virtual slide". The virtual slide
is added to the-total number of slides so that the index time
period assigned to the virtual slide may be used to move the slides
or processing stations back to the starting position. Thus accurate
and predictable incubation times are maintained.
[0056] While a preferred embodiment of the invention has been
described, the invention is susceptible to-modification. For
example, instead of using one or a pair of transfer syringes on an
overhead arm, the reagent carousel could carry a plurality of
micro-delivery reagent fluid dispensers such as described in U.S.
Pat. Nos. 5,232,664 or 5,654,200 or 6,093,574 or 6,192,945.
Moreover, while the use of individually heated thermal platforms is
preferred, the slides may be heated using conventional convection
heating techniques. Still other changes may be made without
departing from the spirit and scope of the invention.
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